Diboron calcium tetraoxide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 June 2020 to 14 July 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EC No 640/2012 Part B. Skin Sensitization: Local Lymph Node Assay, July 2012

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Cas Number:

13701-64-9

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: E00616-912
- Expiration date of the lot/batch: 18 September 2020
- Purity: 82 %

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Young adult animals, approximately 10 weeks old
- Weight at study initiation: 22.5 to 27.5 g
- Housing: Animals were group housed, up to 5 animals of the same sex and same dosing group together, in polycarbonate cages (Makrolon MIII type; height 18 cm) containing sterilised wooden fibres as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study
- Water (e.g. ad libitum): Municipal tap-water was freely available to each animal via water bottles
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 to 23°C
- Humidity (%): 47 to 76 %
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation)
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle
- IN-LIFE DATES: 22 June 2020 to 9 August 2020

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

0, 10, 25 and 40 %

No. of animals per dose:

5

Details on study design:

PREPARATION OF TEST ITEM:
Test item dosing formulations (w/w) were homogenised to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily and dosed within 4 hours after adding the vehicle to the test item. The dosing formulations were kept at room temperature until dosing. The dosing formulations were stirred until and during dosing.

PRE-SCREEN TESTS:
Two test item concentrations were tested; a 25% and 40% concentration. The highest concentration was the highest concentration that could be prepared homogeneously. The test system, procedures and techniques were identical to those used in the main study except that both concentrations were applied using a pipette and that the assessment of lymph node proliferation and necropsy were not performed. Two young adult females per concentration were selected. Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 14 cm). Ear thickness measurements were conducted using a digital thickness gauge (Kroeplin C110T-K) prior to dosing on Days 1 and 3, and on Day 6. Animals were sacrificed after the final observation.

MAIN STUDY :
Three groups (10, 25 and 40 % test item concentrations) of five animals were treated with one test item concentration per group. The highest test item concentration was selected from the pre-screen test. One group of five animals was treated with the vehicle.

Induction of the test item was conducted on days 1, 2 and 3. The dorsal surface of both ears was topically treated (25 µL/ear) with the test item, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing. The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item.

Excision of the nodes was conducted on day 6. Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 µCi of 3H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US). After five hours, all animals were euthanised according to laboratories Standard Operating Procedures. The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in PBS.

In addition, tissue processing for radioactivity was conducted on day 6. Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (maze size: 200 µm, diameter: ± 1.5 cm). LNC were washed twice with an excess of PBS by centrifugation at 200 g for 10 minutes at 4 ºC. To precipitate the DNA, the LNC were exposed to 5 % trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) and then stored in the refrigerator until the next day.

On day 7 radioactivity measurements were taken. Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter (2910TR). Counting time was to a statistical precision of ± 0.2 % or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

Post-dose clinical observations were performed once daily on days 1-6 (on days 1-3 between 3 and 4 hours after dosing). All the animals were examined for reaction to dosing. The onset, intensity and duration of these signs was recorded (if appropriate), particular attention being paid to the animals during and for the first hour after dosing.

Animals were weighed individually on day 1 (pre-dose) and 6 (prior to necropsy).

Erythema and eschar formation observations were performed once daily on days 1-6 (on days 1-3 within 1 hour after dosing), according to the following numerical scoring system. Furthermore, a description of all other (local) effects was recorded.

Erythema and eschar formation:
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema (beet redness) to slight eschar formation (injuries in depth) 3
Severe erythema (beet redness) to eschar formation preventing grading of erythema 4

No necropsy was performed, since all animals survived until the end of the observation period.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

The EC3 value (the estimated item concentration that will give a SI=3) may be determined if possible, based on the dose response relationship or calculated using linear interpolation.

Results and discussion

Positive control results:

The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.5

Test group / Remarks:

10 %

Results 2

Key result

Parameter:

SI

Value:

2.1

Test group / Remarks:

25 %

Results 3

Key result

Parameter:

SI

Value:

1.8

Test group / Remarks:

40 %

Any other information on results incl. tables

PRE-SCREEN TEST:

At a 25 % and 40 % test item concentration, no signs of systemic toxicity were noted and no irritation was observed. Therefore, a 40 % concentration was selected as highest concentration for the main study.

MAIN STUDY:

- Skin Reactions/Irritation:

No irritation was observed in any of the animals. White test item remnants were present on the dorsal surface of the ears of all test item treated animals on days 1-3, which did not hamper scoring of the skin reactions.

- Systemic Toxicity:

No mortality occurred and no clinical signs of systemic toxicity were observed in the animals. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

- Macroscopic Examination of the Lymph Nodes and Surrounding Area:

All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

- Radioactivity Measurements and SI Values:

Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 40 % were 572, 793 and 674 DPM, respectively. The mean DPM/animal value for the vehicle control group was 375 DPM. The SI values calculated for the test item concentrations 10, 25 and 40 % were 1.5, 2.1 and 1.8, respectively.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Since there was no indication that the test item elicits a SI = 3 when tested up to 40 %, the test item was considered not to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 40 %.

The six-month reliability check with alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.

Based on these results, the test item would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Executive summary:

The objective of this study was to evaluate whether the test item induces skin sensitization in mice after three epidermal exposures of the animals under the conditions described in this report.

The study was carried out based on the guidelines described in:

• OECD, Section 4, Health Effects, No.429 (2010).

• EC No 640/2012, Part B: "Skin Sensitization: Local Lymph Node Assay".

• EPA, OPPTS 870.2600 (2003) “Skin Sensitization”.

The six-month reliability check with alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.

Test item concentrations selected for the main study were based on the results of a pre-screen test. Based on the results, the highest concentration required according to the guidelines was selected.

In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 10, 25 or 40 % w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Acetone/Olive oil (4:1 v/v)). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 40 % were 572, 793 and 674 DPM, respectively. The mean DPM/animal value for the vehicle control group was 375 DPM. The SI values calculated for the test item concentrations 10, 25 and 40 % were 1.5, 2.1 and 1.8, respectively.

Since there was no indication that the test item elicits a SI = 3 when tested up to 40 %, the test item was considered not to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 40 %.

Based on these results, the test item would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).